Vitrification of plants is a serious problem since it can affect shoot multiplication and culture vigor and can impede the successful transfer of micropropagated plants to in vivo conditions thereby limiting the application of the in vitro techniques as a means for mass propagation.
Affected plants exhibit tissue hyperhydricity and hypertrophy, deficiency of chlorophyll a and b, cell wall lignification lacking or limited to a layer opposed to the cell membrane, and leaves with large intercellular spaces in the spongy mesophyll, and with little differentiation between the mesophyll and palisade cells (Pasqualetto P. L., W. P. Wergin and R. H. Zimmerman, 1988, Changes in structure and elemental composition of vitrified leaves of `Gala` apple in vitro, Acta Horticulturae 227: 352-367).
Some hypotheses have been proposed for the mechanism of vitrification, and there have been techniques developed to overcome vitrification by manipulating the culture conditions. However, most of the efforts have only limited success and a specific technique usually could not be applied to all plants.
For example, substituting Bacto-agar for Gelrite as a gelling agent could prevent vitrification in `Gala` apple (Pasqualetto P. L., Zimmerman R. H. & Fordham I., 1986, The influence of gelling agent and growth regulator concentrations on vitrification of apple cultivars in vitro, J. Amer. Soc. Hort. Sci. 111: 976-980). For some other plants, however, vitrification could not be prevented by using agar as a gelling agent although it could be decreased to some extent by increasing the concentration of agar. Another example is that removal of cytokinin from the culture medium could reverse the vitrification in melon (Leshem B., Wreker E. & Shalev P. D., 1988, The effect of cytokinins on vitrification in melon and carnation. Ann Bot. 62: 271-276) and apple (Gaspar T., Kever C., Devergh P., Maene L., Pasques M. & Boxus P., 1987, Vitrification: morphological, physiological and ecological aspects, In: Bonga J. M., Durzan D. J., Eds, Cell and Tissue Culture in Forestry, Vol I pp.152-166 Martinus Nijhoff Publ, Dordrecht, Holland) while in cactus it showed no effect. In multiplication cultures of Castanea sativa, normal shoots and vitreous shoots were obtained when using Heller's or MS macronutrient, respectively (Pasqualetto P. L., 1990, Vitrification in plant tissue culture, In: Rodrigues R et al., Eds, Plant aging: Basic and Applied Approaches, pp.133-137, Plenum Press, New York). However, in globe artichoke, changes in the formulation of the major elements did not influence the result of vitrification. Besides, decreased vitrification is usually accompanied by a lower number of adventitious bud regenerations. Therefore, there are still needs for progress in controlling vitrification of plant.
Lettuce is an important vegetable and its in vitro culture is relatively easy. It has been used as a model plant for developing the artificial seeds of adventitious shoots. However, vitrification has proved to be a serious problem after a series of subcultures.
To solve the vitrification problem associated with lettuce cultures, we have tried to manipulate many factors which have been reported effective in limiting vitrification. These factors include culture temperature and concentrations of Ca.sup.2+, NH.sub.4.sup.+, cytokinin, sucrose and polyethyleneglycol. We have also tried two commercial antivitrifying agents (EM1 and EM2, Pronatec Co., France). None of them showed any effect in decreasing vitrification of regenerated shoots.
We surprisingly found that the replacement of agar in a solid culture with water absorbent polymer can minimize vitrification of lettuce without significantly reducing shoot regeneration.